Purpose:
The glial cell line-derived neurotrophic factor (GDNF) is an important trophic factor for multiple types of neurons and glial cells and it’s family members have since been identified as GDNF (binds GFRα1), neurturin (NRTN binds GFRα2), and artemin (ARTN binds GFRα3). To elucidate a role of GDNF receptor-related genes to axonal damage-induced retinal ganglion cell (RGC) death and regeneration, we performed in vitro and vivo experiments with adult mammal retinas.

Methods:
In this study, Sprague-Dawley rats, 200-240 g were used. Firstly, we examined the changes in the expression of GDNF receptors after optic nerve axotomy and then examined neuroprotective effects of GDNF, NRTN, and ARTN on axotomized RGCs in vivo and vitro. Each GDNF family members were intravitreally injected immediate after optic nerve axotomy. At 12 days, the density of surviving RGCs labeled with Fluorogold (FG) was calculated with dissected retinas. In adult primary culture, β3 tubulin-positive surviving RGCs were counted after application of each GDNF family member at specified concentrations. Next, we examined the axon-regenerating effects of each GDNF family members in vitro and vivo. In adult primary culture, the number of β3 tubulin-positive RGCs with elongated axons, more than double RGC soma diameters, were counted. In vivo, each GDNF family member was intravitreally injected after optic nerve crush. Regenerated axons labeled with cholera toxin subunit B were counted at 250, 500 and 1000µm from the crush site with sections of optic nerve.

Results:
We found that optic nerve axotomy induced the upregulation of GFRα3 mRNA level (10-fold) in the retina within 3 days (p<0.01). The density of FG-labeled RGCs was found to be 415 ± 50 cells/ mm2 after injection with PBS, 1013 ± 150 with GDNF (p<0.0001), 1143 ± 150 with NRTN (p<0.0001), and 935 ± 107 with ARTN (p=0.001). Similarly, the application of all GDNF family members (more than 10ng/ml) significantly increased the number of β3 tubulin-positive surviving RGCs in vitro. By contrast, regarding axon-regenerating effects, only application of ARTN had a strong effect on axon regeneration in vitro and vivo, whereas GDNF and NRTN did not.

Conclusions:
Our data suggest GDNF family proteins have a substantial neuroprotective effect on RGCs, and that ARTN has unique effects on axon regeneration.